CN110354144B - Sea squirt extract rich in various plasmalogens and preparation and analysis method thereof - Google Patents
Sea squirt extract rich in various plasmalogens and preparation and analysis method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/616—Echinodermata, e.g. starfish, sea cucumbers or sea urchins
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Abstract
The invention discloses an ascidian extract rich in various plasmalogens and a preparation and analysis method thereof. The method adopts cheap ascidians as raw materials, adopts a certain proportion of water mixed with organic solvent for extraction, and recovers residual plasmalogens in the supernatant, thereby obviously increasing the extraction efficiency and improving the extraction amount of plasmalogens; before high performance liquid phase analysis, hydrochloric acid is used for acidification for 5min to analyze the content of plasmalogen in the extract, and mass spectrum analysis is adopted to analyze the type of the plasmalogen, so that the extract prepared by the method comprises various plasmalogen components and polyunsaturated fatty acids DHA and EPA. The ascidian extract rich in various plasmalogens prepared by the method can be used as a raw material for the fields of nutritional food, health care products related to the prevention and treatment of aging diseases, medicine development and the like.
Description
Technical Field
The invention belongs to the field of food chemistry and biomedicine, and particularly relates to a sea squirt extract rich in multiple plasmalogens and a preparation and analysis method thereof.
Background
Plasmalogens are a specific class of phospholipids, represented by about 10% of the total phospholipids of cells, and are glycerophospholipids containing an olefinic ether bond, such as plasmalogen ethanolamine, plasmalogen phosphatidylcholine, plasmalogen serine, etc. Is mainly characterized by containing an alkenyl ether bond at the sn-1 position and is usually connected with c16:0 in mammals; 18:0 or 18:1 fatty alcohols, polyunsaturated fatty acids which are mostly n-3 and n-6 on sn-2. It is abundant in brain, heart and other organs. Most of the plasmalogens are intensively distributed in a biological membrane lipid raft structure to participate in cell fusion, ion transport and cholesterol transportation, and can be used as an important antioxidant on a plasma membrane to avoid damage of nerve cells.
The results of many studies to date have shown that a decrease in plasmalogen content is associated with a number of diseases such as: alzheimer's disease, Parkinson's disease, Zellweger syndrome, etc. Of these, Alzheimer's Disease (AD) is a progressive neurodegenerative disease, the most common dementia. It was further found that there was a positive correlation between the severity of alzheimer's disease and the degree of reduction in plasmalogens. Therefore, a plurality of animal experiments and clinical experiments are carried out to confirm that the plasmalogen can improve the learning ability and the memory of patients with mild cognitive impairment and mild Alzheimer's disease and mouse models.
Ascidians (Ascidians) belong to ascidian class (ascidiaceae) of phylum chordata (Chordates) and subdivision urochorrdata (urochorrdata) and grow on pearl oyster cages attached to natural sea areas, about 1600 species are grown all over the world and mainly distributed in tropical and subtropical sea areas, Ascidians are widely distributed in China and rich in resources, more than 100 species of recorded coastal Ascidians in China are recorded, and many unique Ascidians in China are animals living in a fixed camp. Compared with terrestrial microorganisms, the ascidian epiphyte can resist a plurality of extreme conditions such as high salt, high pressure, low oxygen, low light and the like which are peculiar to the ocean, thereby forming unique metabolic and physiological characteristics, generating metabolites with different chemical structures, providing active metabolites which can not be provided by the terrestrial microorganisms for human beings, and being unique to the animal world. Therefore, the method is called as the future of biological research and has good application prospect.
At present, some other marine organisms also have plasmalogens, and the Chinese patent publication No. CN108276438A discloses the preparation of EPA plasmalogens by extracting sea cucumber. The EPA plasmalogen is prepared by phospholipase D, and further the plasmalogen phosphatidylcholine (pPC) and the plasmalogen ethanolamine (pPE) are obtained by component separation. However, the invention only extracts EPA plasmalogens singly, does not describe the content of EPA plasmalogens in sea cucumbers, and does not describe other plasmalogens and the separation and content determination.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, it is an object of the present invention to provide an ascidian extract rich in various plasmalogens and methods for preparing and analyzing the same. The invention uses ascidians to prepare ascidian extract rich in various plasmalogens, the lipid metabolite species of ascidians are different from terrestrial organisms, and the ascidian extract contains more abundant phospholipid compounds, so that the plasmalogens compounds can be obtained more efficiently.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing sea squirt extract rich in various plasmalogens comprises the following steps:
(1) preparing fresh sea squirts and carrying out pretreatment; pretreating fresh Halocynthia Roretzi, squeezing out water, cleaning sea mud, removing inedible tissue such as viscera, cutting into small pieces, freezing at-80 deg.C, grinding to obtain homogenate, and freeze drying to obtain lyophilized powder. In order to prevent the oxidation inactivation of plasmalogen components in the subsequent extraction steps, the ascidian freeze-dried powder and antioxidant VE dry powder (the proportion is 1:1% -2%) are uniformly mixed.
(2) Extracting with organic solvent, and vacuum concentrating to obtain ascidian extract rich in plasmalogen:
(a) weighing a certain amount of ascidian freeze-dried powder prepared in the step (1), adding a small amount of water for dilution, adding chloroform-methanol (2:1, v/v), shaking up and shaking, standing at room temperature for 1-2 hours to allow the solvent to stratify; then, centrifugation (6000rpm, 15min) was carried out, and the supernatant was transferred and collected to obtain a residue;
(b) adding chloroform-methanol-water (2:1:0.8, v/v/v), shaking, centrifuging (6000rpm, 15min), transferring and collecting supernatant;
(c) mixing the supernatants of the step (a) and the step (b), adding chloroform and water in a volume ratio of 1:1, shaking and centrifuging; collecting the chloroform phase at the bottom of the container, blowing to dry under nitrogen, vacuum concentrating to obtain ascidian plasmalogen extract, and storing at-20 deg.C.
In the above technical scheme, further, the ascidian extract rich in various plasmalogens is prepared by the above method.
Further, the ascidian extract rich in various plasmalogens comprises 9 phosphatidylethanolamine plasmalogens and 1 phosphatidylserine plasmalogens and plasmalogen components of polyunsaturated fatty acids DHA and EPA.
The invention also provides an analytical method of the ascidian extract rich in various plasmalogens, and the analytical method of the composition and the content of the ascidian extract comprises the following steps:
before and after the sample is treated by hydrochloric acid (the mass fraction of the hydrochloric acid is 36-38%) for 5min, analyzing the content by using HPLC (high performance liquid chromatography); the composition of the ascidian extract was analyzed by mass spectrometry. The plasmalogen compound accounts for a relatively high proportion of the corresponding phospholipid compound and is contained in an amount of about 1.70mg/g dry powder weight; the phosphatidyl ethanolamine plasmalogen (pPE) with 9 different molecular weights was detected in ascidian extract, and its positive ion-to-nucleus ratio [ M + H ]]+Respectively as follows: 776.5(p18:0/22: 6); 752.5(p18:0/20: 4); 750.5(p18:0/20: 5); 716.5(p18:0/17: 1); 748.5(p16:0/22: 6); 722.5(p16:0/20: 5); 746.5(p18:2/20: 5); 784.5(p18:0/22: 2); 798.5(p20:1/22: 6). The values in parentheses indicate the side chain composition of pPE, 22:6 indicates DHA (docosahexaenoic acid) and 20:5 indicates EPA (eicosapentaenoic acid). In addition, 1 phosphatidylserine plasmalogens (pPS) with a positive ion-to-nucleus ratio [ M + H ] was detected]+Is 826.5(p18:0/22: 2).
Furthermore, the ascidian extract rich in various plasmalogens can be used as a raw material for the fields of nutritional foods, health-care products related to the prevention and treatment of aging diseases, drug development and the like.
The invention has the beneficial effects that:
compared with sea cucumbers and other marine organisms, the sea squirts are low in price and low in culture cost, the sea squirts extract comprises various plasmalogen components, and the sea squirts have great advantages as a plasmalogen compound preparation source; according to the invention, a certain proportion of water mixed with an organic solvent is adopted for extraction, and the plasmalogens remained in the supernatant are recovered, so that the extraction efficiency is obviously increased, and the extraction amount of the plasmalogens is increased; the content of plasmalogen in the extract is analyzed by hydrochloric acid acidification (mass fraction is 36-38%) for 5min before high performance liquid phase analysis, and the type of plasmalogen is analyzed by mass spectrometry, so that the extract prepared by the method comprises various plasmalogen components and polyunsaturated fatty acids DHA and EPA. The ascidian extract rich in various plasmalogens prepared by the method can be used as a raw material for the fields of nutritional foods, health-care products related to the prevention and treatment of aging diseases, medicine development and the like.
Drawings
FIG. 1 is a liquid chromatogram of a standard PE.
FIG. 2 is a chromatogram of a mixture of PE and PS as standard substances.
FIG. 3 is a chromatogram of an ascidian phospholipid assay.
Figure 4 is a chromatogram of an assay of ascidianin after acidification for 3 minutes in hydrochloric acid fumes.
Figure 5 is a chromatogram of an assay of ascidianin after acidification for 5 minutes in hydrochloric acid fumes.
FIG. 6 is a mass spectrometric analysis of the composition of ascidianin.
The specific implementation method comprises the following steps:
a method for preparing sea squirt extract rich in various plasmalogens comprises the following steps:
(1) sample pretreatment: the ascidian sample is from Halocynthia roretzi. Taking 10g of a fresh sample after pretreatment, wherein the pretreatment process comprises squeezing out water, cleaning sea mud, removing inedible tissues such as internal organs and the like; cutting into small pieces with scissors, freezing at a temperature below-80 deg.C, grinding in a mortar to obtain homogenate, freezing at-80 deg.C, and lyophilizing with a lyophilizer to obtain about 200mg of lyophilized powder.
(2) Preparation of ascidian extract rich in plasmalogens:
(a) taking about 100mg of the freeze-dried powder, and diluting the freeze-dried powder to 2ml with water;
(b) the suspension is put into a 25ml glass centrifuge tube (centrifuge tube 1), 7.5ml chloroform-methanol (2:1, v/v) is added, shaking is carried out, and the suspension is placed for about 1 hour at room temperature to lead the solvent to be layered;
(c) centrifugation was carried out at 6000rpm for 15 min. After centrifugation, the supernatant was transferred to another 25ml glass centrifuge tube (centrifuge tube 2);
(d) adding 9.5ml chloroform-methanol-water (2:1:0.8, v/v/v) into the centrifuge tube 1, shaking up, and centrifuging at 6000rpm for 15 min;
(e) mixing the two supernatants, adding 5ml of chloroform and 5ml of water, shaking, and centrifuging;
(f) the chloroform phase at the bottom was taken out into a glass vial with a cover, blown dry under nitrogen, concentrated under vacuum and stored in a refrigerator at-20 ℃.
(a-f) the volume of the preparation process can be enlarged.
(3) High performance liquid chromatography analysis of plasmalogen extract
(a) High performance liquid chromatography conditions
A chromatographic column: tnatural C18Column (5um 4.6X 250 mm); the mobile phase is n-hexane, isopropanol and a 1.7% phosphoric acid aqueous solution which is a solution of 45:48:7(v/v/v), wherein the phosphoric acid aqueous solution is a mixture of water and 85% phosphoric acid with the ratio of 50:1(v/v), the flow rate is 1ml/min, and the detection wavelength is 205 nm.
(b) Preparation of Standard and sample solutions
Phosphatidylethanolamine (PE) and Phosphatidylserine (PS) standards (Sigma) are weighed accurately to obtain 5mg of each standard, diluted with n-hexane and added to 5ml of volume to prepare a standard solution with the concentration of 1 mg/ml. A quantity of sample was weighed in triplicate, one for normal ascidianin extract, a second for ascidianin extract acidified for 3 minutes in concentrated hydrochloric acid fumes and a third for 5 minutes in concentrated hydrochloric acid fumes and diluted to a certain concentration with n-hexane. The aim of hydrochloric acid acidification is to carry out acidolysis on plasmalogen to form products such as detectable hemolytic phospholipid, and the like, and the specific method is to drop 5 drops of hydrochloric acid into a test tube cover, invert the test tube containing the sample in hydrochloric acid smoke for processing and timing;
(c) high performance liquid chromatogram analysis
FIG. 1 is a chromatogram result chart of a standard substance in a mobile phase n-hexane/isopropanol/water system. It can be seen that PE peaks relatively early, approximately around two minutes.
FIG. 2 shows the chromatographic results of the standard in a mobile phase n-hexane/isopropanol/phosphoric acid aqueous solution system, the peak of the standard mixed solution is obvious, and the PE retention time is 2.2min and is earlier than PS (3 min).
In FIG. 3, the liquid chromatogram of the sample dissolved in n-hexane showed that the PE peak was detected and the PS peak (lower content) was also observed at retention time (3.7 min). The phospholipid content of the other two peaks in the ascidian extract was not characterized, since no other standards were used as controls.
In the experiment of fig. 4, we performed a chromatogram of a 3 minute acid hydrolysis of a sample under a mist of concentrated hydrochloric acid, and observed no significant difference from the original sample, indicating that plasmalogen is stable for a limited acid hydrolysis time for the ascidian extract.
Fig. 5 shows the results observed after 5 minutes of acidolysis of the sample, and the phosphatidylethanolamine plasmalogen (pPE) is acidified by hydrochloric acid to the corresponding hemolytic phospholipid (lyso-PE), and by calculating the ratio of the chromatographic peak areas, it can be found that the pPE is about 5:1, the plasmalogen compounds account for a higher proportion of the corresponding phospholipid compounds, and the plasmalogen content is about 1.70mg/g dry powder weight.
(4) Mass spectrometry of sphingomyelin extract plasmalogen composition
(a) Mass spectrometry conditions:
the instrument used a Maxis QTOF mass spectrometer (Bruker Daltonics, Billerica, MA USA); ACQUITY UPLC liquid chromatograph (Waters, USA). Mass spectrum conditions: positive ion MS scanning, m/z 300-. Chromatographic conditions are as follows: BEH C18column (2.1 mm. times.100 mm,1.7 μm) (Waters Corp., Milford, USA); mobile phase: the phase A is water, and the phase B is methanol. The gradient elution procedure was: 3% B (0.0-0.5 min); 3-80% B (0.5-8.0 min); 80-100% B (8.0-14.0 min); 100% B (14.0-19.0 min); 100-3% B (19.0-19.1 min); 3% B (19.1-22.0 min). Column temperature: 30 ℃; the flow rate was 0.3ml/min and the amount of sample was 1. mu.l. (b) Mass spectrogram analysis result
FIG. 6 shows the results of mass spectrometry for the composition of plasmalogen. 9 kinds of phosphatidylethanolamine plasmalogens (pPE) with different molecular weights were detected in the ascidian extract, and their positive ions [ M + H ]]+Respectively as follows: 776.5(p18:0/22: 6); 752.5(p18:0/20: 4); 750.5(p18:0/20: 5); 716.5(p18:0/17:1) (ii) a 748.5(p16:0/22: 6); 722.5(p16:0/20: 5); 746.5(p18:2/20: 5); 784.5(p18:0/22: 2); 798.5(p20:1/22: 6). The values in parentheses indicate the side chain composition of pPE, 22:6 indicates DHA (docosahexaenoic acid) and 20:5 indicates EPA (eicosapentaenoic acid). In addition, phosphatidylserine plasmalogens (pPS) were detected, the positive ion of which [ M + H ]]+Is 826.5(p18:0/22: 2). The corresponding graph of each pPE component is: fig. 6.1, fig. 6.2, fig. 6.3, fig. 6.4, fig. 6.5, fig. 6.6, fig. 6.7, fig. 6.8, fig. 6.9. FIG. 6.10 shows pPS.
Claims (2)
1. A method for preparing sea squirt extract rich in multiple plasmalogens is characterized in that the sea squirt extract comprises 9 phosphatidylethanolamine plasmalogens, 1 phosphatidylserine plasmalogens and plasmalogen components of polyunsaturated fatty acids DHA and EPA; the preparation method comprises the following steps:
(1) preparing fresh Halocynthia Roretzi, and pretreating: pretreating fresh Halocynthia Roretzi comprises squeezing out water, cleaning sea mud, removing viscera tissue, cutting into uniform small pieces, freezing at-80 deg.C, grinding to obtain homogenate, freeze-drying to obtain lyophilized powder of Halocynthia Roretzi, and mixing with antioxidant VE dry powder at mass ratio of 1:1% -2%;
(2) extracting with organic solvent, and vacuum concentrating to obtain ascidian extract rich in plasmalogen:
(a) adding water accounting for 2% -2.5% of the mass of the sea squirt freeze-dried powder into the sea squirt freeze-dried powder mixed with the antioxidant VE dry powder, diluting and shaking up, adding chloroform and methanol in a volume ratio of 2:1, continuing shaking up, standing at room temperature for 1-2 hours to stratify a solvent, then centrifuging at 6000rpm for 15min, transferring and collecting supernatant to obtain a residue;
(b) adding chloroform, methanol and water at volume ratio of 2:1:0.8 into the residue, shaking, centrifuging at 6000rpm for 15min, transferring, and collecting supernatant;
(c) mixing the supernatants of the step (a) and the step (b), adding chloroform and water in a volume ratio of 1:1, shaking and centrifuging; blowing the chloroform phase at the bottom under nitrogen, and vacuum concentrating to obtain ascidian extract rich in plasmalogen.
2. The method for preparing the ascidian extract enriched in the plurality of plasmalogens as claimed in claim 1, wherein the ascidian extract enriched in the plasmalogens is subjected to content and type analysis by high performance liquid chromatography and mass spectrometry, respectively, and the high performance liquid chromatography is used for acidifying the ascidian extract with hydrochloric acid with a mass fraction of 36% -38% for 5min before analyzing the content.
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